Targeting the cAMP and Transforming Growth Factor-β Pathway Increases Proliferation to Promote Re-Epithelialization of Human Stem Cell-Derived Retinal Pigment Epithelium

Abstract

Retinal pigment epithelium (RPE) cell integrity is critical to the maintenance of retinal function. Many retinopathies such as age-related macular degeneration (AMD) are caused by the degeneration or malfunction of the RPE cell layer. Replacement of diseased RPE with healthy, stem cell-derived RPE is a potential therapeutic strategy for treating AMD. Human embryonic stem cells (hESCs) differentiated into RPE progeny have the potential to provide an unlimited supply of cells for transplantation, but challenges around scalability and efficiency of the differentiation process still remain. Using hESC-derived RPE as a cellular model, we sought to understand mechanisms that could be modulated to increase RPE yield after differentiation. We show that RPE epithelialization is a density-dependent process, and cells seeded at low density fail to epithelialize. We demonstrate that activation of the cAMP pathway increases proliferation of dissociated RPE in culture, in part through inhibition of transforming growth factor-b (TGF-b) signaling. This results in enhanced uptake of epithelial identity, even in cultures seeded at low density. In line with these findings, targeted manipulation of the TGF-b pathway with small molecules produces an increase in efficiency of RPE re-epithelialization. Taken together, these data highlight mechanisms that promote epithelial fate acquisition in stem cellderived RPE. Modulation of these pathways has the potential to favorably impact scalability and clinical translation of hESC-derived RPE as a cell therapy. STEM CELLS TRANSLATIONAL MEDICINE 2016;5: 925-937 SIGNIFICANCE Stem cell-derived retinal pigment epithelium (RPE) is currently being evaluated as a cell-replacement therapy for macular degeneration. This work shows that the process of generating RPE in vitro is regulated by the cAMP and transforming growth factor-b signaling pathway. Modulation of these pathways by small molecules, as identified by phenotypic screening, leads to an increased efficiency of generating RPE cells with a higher yield. This can have a potential impact on manufacturing transplantation-ready cells at large scale and is advantageous for clinical studies using this approach in the future.

The Controversial Role of TGF-β in Neovascular Age-Related Macular Degeneration Pathogenesis

“…SNAI1 also mediated the increase of fibronectin and α-smooth muscle actin expression, and, consequently, the migratory activity of RPE cells [ 88 ]. As further confirmation of this, it was reported that TGF-β1 led to an increase in expression of mesenchymal markers in stem cell-derived RPE cells, along with a decrease in expression of epithelial markers [ 89 ], and TGF-β2 promoted ARPE-19 cell invasion into collagen by mediating the expression urokinase-type plasminogen activator, a serine protease involved in tissue remodeling and cell migration [ 90 ]. Nevertheless, TGF-β2 was unable to initiate EMT in primary porcine RPE isolated as sheets, cultured in vitro on lens capsules, and as such, maintaining cell–cell contact [ 91 ].…”

Quantified F-Actin Morphology Is Predictive of Phagocytic Capacity of Stem Cell-Derived Retinal Pigment Epithelium

“…Like high-phagocytic RPE cells, low-phagocytic RPE cells lacked the mesenchymal marker protein αSMA and retained localization of the tight junction associated protein ZO-1 at cell-cell contacts. These results suggest that low-phagocytic RPE cells redifferentiated into an epithelial phenotype during the post-proliferative phase in culture ( Choudhary et al., 2016 ). Furthermore, they imply that scoring F-actin organization as described provides information of RPE-like characteristics beyond testing epithelialization.…”

Identification and analysis of long non-coding RNA related miRNA sponge regulatory network in bladder urothelial carcinoma

“…Of these, the neuroactive ligand-receptor interaction signaling pathway has been reported to be associated with the progression of renal cell carcinoma in bioinformatics studies [50]. In addition, previous studies have suggested that cAMP-related signaling could control apoptosis induction and cell growth [51, 52], while another study demonstrated that cAMP was an inhibitor of cell cycle progression and apoptosis in gastric cancer cells [53]. Zhang et al [54] showed that the ECM–receptor interaction pathway played a significant role in tumor progression and metastasis.…”